Hair cortisol level as a molecular biomarker in retinitis pigmentosa patients.

PURPOSE: Retinitis pigmentosa (RP) patients commonly experience negative psychological states due to their progressive and unpredictable loss of vision and visual variations related to stress. The aim of this study was to examine hair cortisol concentrations (HCCs), which is usually associated with chronic stress, pretending to unveil possible associations between underlying psychological factors and disease severity in RP patients. METHODS: Seventy-eight RP patients and 148 healthy controls were included in this study. A complete ophthalmological exam was performed in all patients to grade into severity disease groups. Perceived stress and trait-anxiety were measured by the State-Trait Anxiety Inventory (STAI) questionnaire. RESULTS: Fifty-two (67%) patients had severe RP and 26 (33%) mild-moderate RP. Fifty-eight (58,9%) patients reported severely levels of stress and 18 (23.,1%) highly levels assessed by STAI questionnaire. RP patients exhibited higher HCCs (500.04 ± 120.99 pg/mg) than in controls (136.17 ± 60.51 pg/mg; p < 0.001). Severe RP patients had significant higher HCCs than mild-moderate patients differing in 274.27 pg/mg (p < 0.001). RP severity grade and perceived anxiety levels in the questionaries were not associated. Group differences were not affected by relevant covariates (age, grade of severity, stress status, and gender). CONCLUSIONS: HCC seems an effective biomarker associated with chronic stress in RP patients. This study shows that HCC in patients with RP are elevated compared to population-based controls, and association between HCC and RP severity was found. Future research is needed to characterize the effect of untreated negative psychological states on progression of the disease if any.

Diagnosis of Multisystem Inflammatory Syndrome in Children by a Whole-Blood Transcriptional Signature.

BACKGROUND: To identify a diagnostic blood transcriptomic signature that distinguishes multisystem inflammatory syndrome in children (MIS-C) from Kawasaki disease (KD), bacterial infections, and viral infections. METHODS: Children presenting with MIS-C to participating hospitals in the United Kingdom and the European Union between April 2020 and April 2021 were prospectively recruited. Whole-blood RNA Sequencing was performed, contrasting the transcriptomes of children with MIS-C (n = 38) to those from children with KD (n = 136), definite bacterial (DB; n = 188) and viral infections (DV; n = 138). Genes significantly differentially expressed (SDE) between MIS-C and comparator groups were identified. Feature selection was used to identify genes that optimally distinguish MIS-C from other diseases, which were subsequently translated into RT-qPCR assays and evaluated in an independent validation set comprising MIS-C (n = 37), KD (n = 19), DB (n = 56), DV (n = 43), and COVID-19 (n = 39). RESULTS: In the discovery set, 5696 genes were SDE between MIS-C and combined comparator disease groups. Five genes were identified as potential MIS-C diagnostic biomarkers (HSPBAP1, VPS37C, TGFB1, MX2, and TRBV11-2), achieving an AUC of 96.8% (95% CI: 94.6%-98.9%) in the discovery set, and were translated into RT-qPCR assays. The RT-qPCR 5-gene signature achieved an AUC of 93.2% (95% CI: 88.3%-97.7%) in the independent validation set when distinguishing MIS-C from KD, DB, and DV. CONCLUSIONS: MIS-C can be distinguished from KD, DB, and DV groups using a 5-gene blood RNA expression signature. The small number of genes in the signature and good performance in both discovery and validation sets should enable the development of a diagnostic test for MIS-C.

The role of asynapsis in human spermatocyte failure.

The basic molecular mechanisms by which chromosomal rearrangements in heterozygous state produce spermatogenic disturbances are poorly understood. Testicular biopsies from five patients - one carrier of a Robertsonian translocation rob t(13;14), two carriers of two different Y-autosome translocations, a t(Y;6) and a t(Y;11), one carrier of a reciprocal translocation t(3;13) and one carrier of a heterochromatin duplication in chromosome 9 - were processed for histopathological analysis, electron microscopy and fluorescent immunolocalization of meiotic proteins. In all the patients, the asynaptic regions during pachytene are labelled by BRCA1 and retained RAD51 foci. The variant histone γ-H2AX is located on the chromatin domains of the asynaptic regions and the XY body. In contrast, these meiotic proteins are absent in those chromosomal segments that are non-homologously synapsed. The present observations on five new cases and a review of recent studies show that the common features shared by all these cases are the abnormal location of some meiotic proteins and the presence of transcriptionally silenced chromatin domains on asynaptic regions. The frequent association of these silenced regions with the XY body and the rescue of spermatocyte viability through non-homologous synapsis are also shared by all these carriers. A passive, random mechanism of clustering of asynaptic regions with the XY body is suggested.

Neonatal sepsis and transient immunodeficiency: Potential for novel immunoglobulin therapies?

Neonates, especially preterm neonates, have the highest risk of sepsis of all age groups. Transient immaturity of the neonatal immune system is an important risk factor. Neonates suffer from hypogammaglobulinemia as nor IgA nor IgM is transferred over the placenta and IgG is only transferred over the placenta late in gestation. In addition, neutrophil numbers and complement function are also decreased. This mini-review focuses on strategies to improve neonatal host-defense. Both clinical and preclinical studies have attempted to boost neonatal immunity to lower the incidence of sepsis and improve outcome. Recent advances in the development of (monoclonal) antibodies show promising results in preclinical studies but have yet to be tested in clinical trials. Strategies to increase complement activity seem efficient in vitro but potential disadvantages such as hyperinflammation have held back further clinical development. Increase of neutrophil numbers has been tested extensively in clinical trials but failed to show improvement in mortality. Future research should focus on clinical applicability of promising new prevention strategies for neonatal sepsis.

Monoclonal antibodies effectively potentiate complement activation and phagocytosis of Staphylococcus epidermidis in neonatal human plasma.

Central line associated bloodstream infections (CLABSI) with Staphylococcus epidermidis are a major cause of morbidity in neonates, who have an increased risk of infection because of their immature immune system. As especially preterm neonates suffer from antibody deficiency, clinical studies into preventive therapies have thus far focused on antibody supplementation with pooled intravenous immunoglobulins from healthy donors (IVIG) but with little success. Here we study the potential of monoclonal antibodies (mAbs) against S. epidermidis to induce phagocytic killing by human neutrophils. Nine different mAbs recognizing Staphylococcal surface components were cloned and expressed as human IgG1s. In binding assays, clones rF1, CR5133 and CR6453 showed the strongest binding to S. epidermidis ATCC14990 and CR5133 and CR6453 bound the majority of clinical isolates from neonatal sepsis (19 out of 20). To study the immune-activating potential of rF1, CR5133 and CR6453, bacteria were opsonized with mAbs in the presence or absence of complement. We observed that activation of the complement system is essential to induce efficient phagocytosis of S. epidermidis. Complement activation and phagocytic killing could be enhanced by Fc-mutations that improve IgG1 hexamerization on cellular surfaces. Finally, we studied the ability of the mAbs to activate complement in r-Hirudin neonatal plasma conditions. We show that classical pathway complement activity in plasma isolated from neonatal cord blood is comparable to adult levels. Furthermore, mAbs could greatly enhance phagocytosis of S. epidermidis in neonatal plasma. Altogether, our findings provide insights that are crucial for optimizing anti-S. epidermidis mAbs as prophylactic agents for neonatal CLABSI.

Reduced adipogenic gene expression in fibroblasts from a patient with type 2 congenital generalized lipodystrophy.

AIMS: Beradinelli-Seip congenital generalized lipodystrophy is a rare autosomal recessive disorder characterized by near-complete absence of adipose tissue, Herculean appearance, insulin resistance, hypoleptinaemia and diabetes mellitus. The aim of this study was to investigate the in vitro effects of pioglitazone on the expression of genes involved in adipogenesis in fibroblasts from a patient with this condition due to a seipin mutation. METHODS: Primary cultures of fibroblasts from the skin of the patient were obtained. Fibroblasts were treated with classic adipose differentiation medium, with and without pioglitazone. Several adipogenes were evaluated by real-time reverse transcriptase-polymerase chain reaction and western blotting. Intracellular localization of prelamin A was studied by immunofluorescence microscopy. RESULTS: The expression of the adipogenic genes PPARG, LPL, LEP and SLC2A4 was reduced in lipodystrophic fibroblasts, while treatment with pioglitazone increased the expression of these genes. Moreover, and unexpectedly, we found an accumulation of farnesylated prelamin A in lipodystrophic fibroblasts. CONCLUSIONS: The process of adipocyte differentiation is compromised in patients with Beradinelli-Seip congenital lipodystrophy owing to diminished expression of the regulatory genes involved, which pioglitazone treatment partially rescues. Prelamin A accumulation establishes a link with other types of familial lipodystrophies, as familial partial lipodystrophy.

Focal spermatogenesis originates in euploid germ cells in classical Klinefelter patients.

BACKGROUND: Klinefelter syndrome is the most frequent chromosome abnormality in human males. This paper aims to investigate the ploidy of meiotic and pre-meiotic germ cells found in spermatogenic foci, and furthermore, the sex chromosome constitution of Sertoli cells which surround these germ cells in non-mosaic Klinefelter patients. METHODS AND RESULTS: A survey of 11 adult patients diagnosed with classical, non-mosaic Klinefelter syndrome who underwent testicular biopsies, showed that six of them had spermatogenesis foci. The topographical study of the biopsies showed that tubuli with germ cells are a minor fraction (8-24%) of all tubuli, although the overwhelming majority is devoid of germ cells. Using fluorescence in situ hybridization (FISH) with probes for the X-centromere and immunolocalization of meiotic proteins, the present work shows that all the 92 meiotic spermatocytes analyzed with FISH were euploid, 46,XY, and thus can form normal, haploid gametes. On the other hand, Sertoli cells show two marks for the X chromosome, meaning that they are 47,XXY. CONCLUSIONS: These results provide a rationale for the high rate of success in the testicular sperm extraction plus ICSI procedures when applied to Klinefelter patients. It is also in agreement with previous studies in the XXY-mouse model. These spermatogenic foci most probably originate from clones of spermatogonia that have randomly lost one of the X chromosomes, probably during periods of life when high spermatogonial mitotic activity occurs.

Effectiveness and safety of nutritional supplements in the treatment of hereditary retinal dystrophies: a systematic review.

The hereditary retinal dystrophies (HRDs) are a group of genetically determined disorders that result in loss of the visual function. There is a lack of standard pharmacological treatments or widely accepted nutritional recommendations. The objective of this review is to summarise the scientific evidence on the effectiveness and safety of nutritional supplements for the treatment of HRDs. We conducted a scientific literature search on Medline and PreMedline, EMBASE, SCI-EXPANDED, SSCI, and The Cochrane Library up to August 2014. Experimental, quasi-experimental and controlled observational studies were selected. Eight studies were ultimately included, seven on retinitis pigmentosa (RP) and one on Best disease. Vitamin A, vitamin E, docosahexaenoic acid (DHA), lutein and ß-carotene were assessed. A 15 000 IU daily dose of vitamin A was reported to have shown a small protective effect on the progression of RP, as was the use of the carotenoids lutein and ß-carotene. Different DHA doses has no effect on RP or Best disease. No supplement showed severe adverse effects in the selected studies although strong evidence of toxicity exists for high doses of vitamin A and ß-carotene in certain populations. The selected studies concluded that there may be a small beneficial effect of vitamin A, lutein and ß-carotene on the progression of RP. The limited evidence available indicates some well-designed additional studies on combined supplements strategies may achieve more robust conclusions. Moreover, the scarcity of evidence available on the treatment of HRD other than RP with nutritional supplements supports the need for further research efforts.

Suicidios en el Perú: Descripción epidemiológica a través del Sistema Informático Nacional de Defunciones (SINADEF) en el periodo 2017-2021 / Suicides in Perú: Epidemiological description through SINADEF in the period 2017-2021

RESUMEN Objetivo: Descripción de características epidemiológicas de suicidios ocurridos en el Perú durante el periodo 2017-2021. Material y Métodos: Estudio de tipo descriptivo-comparativo, basado en la revisión del Sistema Informático Nacional de Defunciones entre los años 2017-2021; la muestra final incluyó un total de 2579 suicidios. Resultados: El 69,5% de los suicidios ocurrieron en el género masculino, el promedio de edad fue 34,87 años, 79,5% solteros y 26,6% con instrucción secundaria completa. Los departamentos con mayor frecuencia de suicidio fueron Lima (20,3%), Arequipa (19,2%), Cusco (9,5%) y Junín (7,8%). Según CIE-10 la causa principal de muerte fue edema cerebral (20,06%) y el domicilio, el lugar más frecuente (63,4%). Las modalidades utilizadas fueron ahorcamiento (56,6%), envenenamiento (29,2%), arma de fuego (4,0%), precipitación (3,1%) y arma blanca (1,7%). Se pudo establecer diferencias significativas con un p < 0,05 entre tipo de suicidio y género, identificando un 74% de casos por ahorcamiento en el sexo masculino; entre tipo de suicidio y región natural: 60% de ahorcamientos en la costa; entre rango etario y tipo de suicidio: ahorcamiento 10-19 años (27,7%) y envenenamiento 20-29 años (26,5%), La mayoría (70,7%) cometió suicidio por ahorcamiento en su domicilio, seguida por envenenamiento en establecimientos de salud (56,9%) y por precipitación en la vía pública (16,8%). Conclusiones: El tipo más frecuente de suicidio fue por ahorcamiento, en personas jóvenes-adultas de sexo masculino y por envenenamiento en personas de sexo femenino.

SUMMARY Objectives: Description of epidemiological features of suicides in Peru between the years 2017-2021. Material and Methods: Descriptive-comparative study based on the review of the national computerized registry of SINADEF deaths between the years 2017-2021; the final sample included a total of 2579 suicides. Results: Male gender reached 69.5%, average age 34.87 years, 79.5% were single, and 26.6% had complete secondary education. The departments with highest frequency of suicides were Lima (20.3%), Arequipa (19.2%), Cusco (9.5%) and Junín (7.8%). According to ICD-10, the main cause of death was cerebral edema (22.71%), and place of death at home (63.4%). Suicide modalities included hanging (56.6%), poisoning (29.2%), firearm (4%), precipitation (3.1%), and knife (1.7%). It was possible to establish significant differences with a p<0.05 value in: type of suicide and gender, 74% of hangings among males; type of suicide and natural region: hanging in the coast (60%); type of suicide and age range: hanging 10-19 years (27.7%) and poisoning 20-29 years (26.5%). A majority (70.7%) died at home by hanging, followed by poisoning in health facilities (56.9%), and by precipitation (16.8%) on public roads. Conclusions: The most frequent type of suicide was by hanging in young-adult males, and poisoning among females.

Intranuclear localization of phospholipids by ultrastructural cytochemistry.

The presence of phospholipids within the interphase nucleus and in isolated chromatin, previously demonstrated by analytical biochemical methods, has been only rarely documented by cytochemical procedures, especially at the ultrastructural level. By means of a gold-conjugated phospholipase technique, we investigated the fine localization of endogenous phospholipids in the different nuclear domains in rat pancreas and in cell cultures. To reduce possible removal or displacement of phospholipids, different specimen preparation procedures such as cryofixation, cryosectioning, and freeze-fracturing were utilized. Apart from slight differences in efficiency among these methods, phospholipids have been cytochemically identified in the same nuclear domains: the interchromatin granules and fibers and the dense fibrillar component of the nucleolus. These results suggest that the phospholipids are an actual nuclear component, not randomly distributed in the nucleoplasm but mainly localized in the nuclear domains involved in the synthesis, maturation, and transport of ribonucleoproteins.

Cytogenetic findings in 200 children with mental retardation and multiple congenital anomalies of unknown cause.

We report a cytogenetic study of 200 children with mental retardation and three or more major or minor congenital anomalies. In all cases, the chromosomes were studied with conventional staining methods (nonbanding) and with at least one of the following techniques: Q, G, or R banding. In a few patients, C banding and in vitro differentiation with BUDR and acridine orange (R banding) were also used. In patients with structural abnormality, parental chromosomes were studied using the same techniques. A chromosomes abnormality was found in 42 patients (21%). Of these, 16(8%) had complete or mosaic aneuploidies (11 autosomal and 5 gonosomal); and 26 (13%) had structural defects. In 21 of the latter the structural abnormality occurred as a de novo rearrangement, and in 5 the defect was inherited from a parent carrier of a balanced rearrangement. The contribution of chromosome aberrations to the cause of (idiopathic) MCA/MR syndromes is discussed.

Brief clinical report: interstitial deletion of the long arm of chromosome 4, del(4)(q28-->q31.3).

Interstitial deletions of the long arm of chromosome 4 are rare. Different breakpoints are involved. Only one of the patients had a very similar deletion to that of the present case. Both had low birth weight at term; weight, length and head circumference less than the third centile; epicanthic folds; apparently low-set abnormal ears; broad nasal bridge; micrognathia; hypoplastic nails; delayed psychomotor development; and mild mental retardation.

High-performance liquid chromatographic determination of vancomycin in rabbit serum, vitreous and aqueous humour after intravitreal injection of the drug.

A high-performance liquid chromatographic method for the determination of vancomycin in rabbit serum, vitreous and aqueous humour has been developed. No clean-up step was necessary for vitreous and aqueous humour samples. For serum samples liquid-liquid and solid-phase extraction were tested and the best results were achieved using C18 cartridges. The extracts were analyzed on a C18 reversed-phase column, using a mixture of 0.05 M phosphate buffer (pH 4) with 10% of acetonitrile as mobile phase. The detection was carried out at 198 nm, which allows higher sensitivity. The average quantitation limit obtained was 0.03 micrograms/ml. The method has been applied to the study of the residual quantities of vancomycin in serum and rabbit eyes after intravitreal administration of the drug in endophthalmitis treatment.

Chromosomal sensitivity to X-rays in lymphocytes from patients with Turner syndrome.

Lymphocytes from patients with Turner syndrome were irradiated with X-rays (200 rad) to determine the chromosomal aberration frequency in first-division metaphases. Five patients with 45,X karyotype; three 45,X/46,Xi(X)q mosaics; one 45,X/47,XXX mosaic and 9 female controls were studied. Patients with a 45,X karyotype exhibited a radioinduced chromosomal aberration frequency similar to controls (38.6 +/- 6.37 and 36.2 +/- 5.11 respectively; p = 0.42). In the mosaics, 45,X cells had a mean frequency of 38.75 +/- 2.16; 46,Xi(X)q cells a mean of 38 +/- 2.16 and the control group a rate of 36.25 +/- 4.32. No differences were observed between 45,X and 46,Xi(X)q cells (p = 0.50), 45,X and normal cells (p = 0.24) or 46,Xi(X)q and normal cells (p = 0.35). Apparently neither the X monosomy nor the Xq isochromosome influences the 'in vitro' X-ray-induced chromosomal damage in Turner syndrome lymphocytes.

X-radiation-induced chromosome breakage in retinoblastoma lymphocytes.

We have examined the spontaneous and X-radiation-induced chromosomal damage in normal humans and in patients with retinoblastoma using the BudR-Giemsa technique in lymphocytes cultured for 48 h. 9 sporadic unilateral non-hereditary cases, 11 hereditary cases (8 bilateral sporadic and 3 unilateral hereditary cases) and 20 healthy individuals were studied simultaneously. No difference in the spontaneous frequency of chromatid and chromosome aberrations was observed between patients and controls. After treatment with 150 rad the frequency of chromosome exchange aberrations was higher in unilateral hereditary cases than the controls (42.0% +/- 5.3 and 22.3% +/- 2.6 respectively; p = 0.05). In bilateral sporadic retinoblastoma 2 different groups were observed. A hypersensitive group showed a significant increment in radiation-induced chromosomal exchange aberrations over the control group (46.2% +/- 5.4 and 24.2% +/- 2.1 respectively; p = 0.01). The other group had a chromosomal exchange frequency similar to normal individuals (26.5% +/- 2.0 and 24.2% +/- 0.4 respectively; p = 0.10). Sporadic unilateral non-hereditary retinoblastoma had an exchange chromosomal aberration frequency similar to control individuals (26.1% +/- 2.8 and 24.6% +/- 2.7 respectively; p greater than 0.10). These results suggest that: There is no relationship between spontaneous chromosome fragility and retinoblastoma. Sporadic unilateral non-hereditary retinoblastoma has normal chromosome sensitivity to X-irradiation. Some hereditary cases of retinoblastoma are sensitive to X-rays while others behave like normals. A mutation or a submicroscopic deletion at a DNA repair locus which is independent of the retinoblastoma gene may cause this radiosensitivity.

Pharmacokinetics of 0.5 mg of a single and a multiple dose of intravitreal vancomycin in infected rabbit eyes.

The purpose of this study was to determine the pharmacokinetics governing distribution and elimination of 0.5 mg of intravitreal vancomycin in a single dose and in a multiple therapeutic regime in infected rabbit eyes. A total of 96 rabbits was injected with approximately 200 CFU of S. aureus intravitreally. Four days later, a single dose of 0.5 mg of vancomycin was administered to Group I (n=36). Group II (n=60) was injected with a maximum of 4 doses of 0.5 mg every 36 hr. Four animals were sacrificed at different time points in each group. Samples of vitreous, aqueous and blood were taken from each animal for analyses by HPLC. These results were evaluated using the RSTRIP program. High vancomycin concentrations were demonstrated in the vitreous of Group I, with a calculated half-life of 12 hr. In Group II, vancomycin levels were within the therapeutic range during the entire experiment. There was minimal accumulation of the drug, and the half-life did not seem to be longer with multiple doses. In conclusion, the pharmacokinetics do not change significantly when a multidose regime is used compared with a single dose. Therapeutic intravitreal concentrations of vancomycin can be achieved by using repeated doses of 0.5 mg of vancomycin.

Pharmacokinetics of intravitreal vancomycin in normal and infected rabbit eyes.

The purpose of this study was to determine the pharmacokinetics governing the distribution and elimination of intravitreally injected vancomycin in normal and infected rabbit eyes. Two groups each of 36 pigmented animals were used. Group 1 served as control. In Group 2, experimental endophthalmitis was induced in the right vitreous by inoculation with Staphylococcus aureus. Once endophthalmitis developed, a vancomycin solution was injected. Four animals from each group were killed at nine time points post-injection, the vitreous and aqueous were removed, and blood samples were taken for HPLC analysis. Data analysis was performed using the RSTRIP program. The half-lives were 69 hours in normal vitreous and 14.53 hours in infected vitreous. Therapeutic drug levels were present in the vitreous 84 hours post-injection in all eyes; they were detected from 2 to 48 hours in normal aqueous but at lower levels in the infected ones. Kv and Ca/Cv ratios suggested that the primary route of elimination was across the retina and the anterior chamber in normal eyes, and via the retina in infected eyes. Results indicate that pharmacokinetic parameters change in pathological conditions, which may help establish better treatment guidelines for endophthalmitis.

A high yield technique for freeze-fracturing of small fractions of isolated cells.

A simple, high-yield technique for the freeze-fracturing of small amounts of isolated cells is described. A drop of cells fixed in suspension is deposited on a polylysine-treated coverslip, forming a monolayer through electrostatic forces. After cryoprotection, the coverslip is inverted on a gold carrier covered with Vinol and then frozen in liquid nitrogen. The monolayer will be fractured by advancing the knife under the coverslip. Large areas of cell surface can be exposed despite their low number, such as that obtainable after cell sorting by flow cytometry.

[Retinal shortening: the most severe form of proliferative vitreoretinopathy (PVR)]. / Acortamiento retiniano: la forma más grave de la vitreorretinopatía proliferante (VRP).

PURPOSE: To identify the clinical characteristics of patients developing retinal shortening due to intraretinal PVR. METHODS: Observational and retrospective cohort study on 110 PVR patients operated on between 2000 and 2001. During surgery, after removing epiretinal membranes and ruling out the presence of subretinal membranes, a perfluorocarbon liquid was injected. Those cases in which retinal flattening was not accomplished, were considered intraretinal PVR (group 1). Those in which retinal flattening allowed endolaser application, were taken as the control group (group 2). Clinical features of both groups were compared by chi-square test. RESULTS: 60 cases (54.5%, CI 95%: 40.5-68.5) showed retinal shortening (group 1). In 24 cases (21.8%, CI 95%: 12.9-30.7) complete retinal flattening was accomplished (group 2). In 26 cases (23.6%), evaluation was inconclusive. In 9 out of the 60 cases of group 1 (15%) a retinectomy was necessary to reattach the retina. Differences between both groups were not statistically significant for any of the clinical variables. However, the number of retinal detachments of more than 60 days of evolution was significantly higher in retinectomized eyes (20.7%) than in group 1 (3.7%) (p=0.04). CONCLUSIONS: Retinal shortening is a relatively frequent phenomenon in PVR. Further studies are necessary to characterize this clinical presentation of PVR and its pathogenesis.